The present invention relates to the manufacture and identification of novel monoclonal antibodies to human B7, i.e., human B7.1 and human B7.2 and primatized forms thereof. More specifically, the present invention relates to the production and identification of macaque antibodies to human B7, i.e., human B7.1 and human B7.2 produced by screening of phage display libraries and monkey heterohybridomas using B lymphocytes obtained from B7 immunized monkeys.
The invention further relates to specific primatized antibodies which bind to human B7, i.e., human B7.1 and B7.2 as well as their corresponding amino acid and nucleic acid sequences.
Also, the present invention relates to pharmaceutical compositions containing monkey monoclonal or primatized antibodies specific to human B7.1 and/or human B7.2 and their use as immunosuppressants by modulating the B7:CD28 pathway, e.g., for the treatment of autoimmune disorders, and the prevention of organ rejection.
The clinical interface between immunology, hematology, and oncology has long been appreciated. Many conditions treated by the hematologist or oncologist have either an autoimmune or immunodeficient component to their pathophysiology that has led to the widespread adoption of immunosuppressive medications by hematologists, whereas oncologists have sought immunologic adjuvants that might enhance endogenous immunity to tumors. To date, these interventions have generally consisted of nonspecific modes of immunosuppression and immune stimulation. In addition to the limited efficacy of these interventions, toxicities secondary to their nonspecificity have also limited their overall success. Therefore, alternative strategies have been sought.
Elucidation of the functional role of a rapidly increasing number of cell surface molecules has contributed greatly to the integration of immunology with clinical hematology and oncology. Nearly 200 cell surface antigens have been identified on cells of the immune and hematopoietic systems (Schlossman S F. Boumsell L. Gilks J M, Harlan T. Kishimoto, C. Morimoto C, Ritz J. Shaw S, Silverstein R L, Springer T A, Tedder T F, Todd R F:CD antigens (1993), Blood 83:879, 1994). These antigens represent both lineage-restricted and more widely distributed molecules involved in a variety of processes, including cellular recognition, adhesion, induction and maintenance of proliferation, cytokine secretion, effector function, and even cell death. Recognition of the functional attributes of these molecules has fostered novel attempts to manipulate the immune response. Although molecules involved in cellular adhesion and antigen-specific recognition have previously been evaluated as targets of therapeutic immunologic intervention, recent attention has focused on a subgroup of cell surface molecules termed co-stimulatory molecules (Bretscher P: xe2x80x9cThe two-signal model of lymphocyte activation twenty-one years later.xe2x80x9d Immunol. Today 13:73, (1992); Jenkins M K, Johnson J G: xe2x80x9cMolecules involved in T-cell co-stimulation.xe2x80x9d Curr Opin Immunol 5:351, 1993; Geppert T, Davis L. Gur H. Wacholtz M. Lipsky P: xe2x80x9cAccessory cell signals involved in T-cell activation.xe2x80x9d Immunol Rev 117:5, (1990); Weaver C T, Unanue E R: xe2x80x9cThe co-stimulatory function of antigen-presenting cells.xe2x80x9d Immunol Today 11:49, (1990); Stennam R M, Young J W: xe2x80x9cSignals arising from antigen-presenting cells.xe2x80x9d Curr Opin Immunol 3:361, (1991)). Co-stimulatory molecules do not initiate but rather enable the generation and amplification of antigen-specific T-cell responses and effector function (Bretscher P: xe2x80x9cThe two-signal model of lymphocyte activation twenty-one years later.xe2x80x9d Immunol. Today 13:73, (1992); Jenkins M K, Johnson J G: xe2x80x9cMolecules involved in T-cell co-stimulation.xe2x80x9d Curr Opin Immunol 5:351, (1993); Geppert T, Davis L. Gur H. Wacholtz M. Lipsky P: xe2x80x9cAccessory cell signals involved in T-cell activation.xe2x80x9d Immunol Rev 117:5, (1990); Weaver C T, Unanue E R: xe2x80x9cThe co-stimulatory function of antigen-presenting cells.xe2x80x9d Immunol Today 11:49, (1990); Stennam R M, Young J W: xe2x80x9cSignals arising from antigen-presenting cells.xe2x80x9d Curr Opin Immunol 3:361, (1991); June C H, Bluestone J A, Linsley P S, Thompson C D: xe2x80x9cRole of the CD28 receptor in T-cell activation.xe2x80x9d Immunol Today 15:321, (1994).
Recently, one specific co-stimulatory pathway termed B7:CD28 has been studied by different research groups because of its significant role in B and T cell activation (June C H, Bluestone J A, Linsley P S, Thompson C D: xe2x80x9cRole of the CD28 receptor in T-cell activation.xe2x80x9d Immunol Today 15:321, (1994); June C H, Ledbetter J A: xe2x80x9cThe role of the CD28 receptor during T-cell responses to antigen.xe2x80x9d Annu Rev Immunol 11:191, (1993); Schwartz R H: xe2x80x9cCo-stimulation of T lymphocytes: The role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.xe2x80x9d Cell 71:1065, (1992)). Since this ligand: receptor pathway was discovered four years ago, a large body of evidence has accumulated suggesting that B7:CD28 interactions represent one of the critical junctures in determining immune reactivity versus anergy (June C H, Bluestone J A, Linsley P S, Thompson C D: xe2x80x9cRole of the CD28 receptor in T-cell activation.xe2x80x9d Immunol Today 15:321, (1994); June C H, Ledbetter J A: xe2x80x9cThe role of the CD28 receptor during T-cell responses to antigen.xe2x80x9d Annu Rev Immunol 11:191, (1993); Schwartz R H: xe2x80x9cCo-stimulation of T lymphocytes: The role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.xe2x80x9d Cell 71:1065, (1992); Cohen J: xe2x80x9cMounting a targeted strike on unwanted immune responsesxe2x80x9d (news; comment). Science 257:751, (1992); Cohen J: xe2x80x9cNew protein steals the show as xe2x80x98co-stimulatorxe2x80x99 of T cellsxe2x80x9d (news; comment). Science 262:844, (1993)).
In particular, the role of the human B7 antigens, i.e., human B7.1 and B7.2, has been reported to play a co-stimulatory role in T-cell activation.
1. B7.1 and B7.2 Co-stimulatory Role in T Cell Activation
The elaboration of a successful immune response depends on a series of specific interactions between a T cell and an antigen presenting cell. Although the essential first step in this process depends upon the binding of antigen to the T cell receptor, in the context of the MHC class II molecule (Lane, P. J. L., F. M. McConnell, G. L. Schieven, E. A. Clark, and J. A. Ledbetter, (1990), xe2x80x9cThe Role of Class II Molecules in Human B Cell Activation.xe2x80x9d The Journal of Immunology, 144:3684-3692), this interaction alone is not sufficient to induce all the events necessary for a sustained response to a given antigen (Schwartz, R. H. (1990), xe2x80x9cA Cell Culture Model for T Lymphocyte Clonal Anergy.xe2x80x9d Science, 248:1349; Jenkins, M. K. (1992). xe2x80x9cThe Role of Cell Division in the Induction of Clonal Anergy.xe2x80x9d Immunology Today, 13:69; Azuma, M., M. Catabyab, D. Buck, J. H. Phillips, and L. L. Lanier, (1992). xe2x80x9cInvolvement of CD28 in MHC-unrestricted Cytotoxicity Mediated by a Human Natural Killer Leukemia Cell Line.xe2x80x9d The Journal of Immunology, 149:1556-1561; Azuma, M., M. Catabyab, D. Buck, J. H. Phillips, and L. L. Lanier, (1992). xe2x80x9cCD28 Interaction with B7 Costimulates Primary Allogeneic Proliferative Responses and Cytotoxicity Mediated by Small Resting T Lymphocytes.xe2x80x9d J. Exp. Med., 175:353-360).
The involvement of certain other co-stimulatory molecules is necessary (Norton, S. D., L. Zuckerman, K. B. Urdahl, R. Shefner, J. Miller, and M. K. Jenkins. (1992), xe2x80x9cThe CD28 Ligand, B7, Enhances IL-2 Production by Providing A Costimulatory Signal to T Cells.xe2x80x9d The Journal of Immunology, 149:1556-1561). xe2x80x9cThe homodimers CD28 and CTLA-4 expressed on T cellsxe2x80x9d (June, C. H., J. A. Ledbetter, P. S. Linsley, and C. B. Thompson, (1990), xe2x80x9cRole of the CD28 Receptor in T-Cell Activation.xe2x80x9d Immunology Today, 11:211-216; Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire, N. K. Damle, and J. A. Ledbetter, (1991), xe2x80x9cCTLA-4 is a Second Receptor for the B Cell Activation Antigen B7.xe2x80x9d J. Exp. Med., 174:561), together with B7.1 (CD80) and B7.2 (CD86) expressed on antigen presenting cells, are major pairs of co-stimulatory molecules necessary for a sustained immune response (Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L. L. Lanier, (1993), xe2x80x9cFunctional Expression of B7/BB1 on Activated T Lymphocytes.xe2x80x9d J. Exp. Med., 177:845-850; Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler, (1989), xe2x80x9cB7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells.xe2x80x9d The Journal of Immunology, 143:2714-2722; Hathcock, K. S., G. Laslo, H. B. Dickler, J. Bradshaw, P. Linsley, and R. J. Hodes, (1993), xe2x80x9cIdentification of an Alternative CTLA-4 Ligand Costimulatory for T Cell Activation.xe2x80x9d Science, 262:905-911; Hart, D. N. J., G. C. Starling, V. L. Calder, and N. S. Fernando, (1993). xe2x80x9cB7/BB-1 is a Leucocyte Differentiation Antigen on Human Dendritic Cells Induced by Activation.xe2x80x9d Immunology, 79:616-620). It can be shown in vitro that the absence of these co-stimulatory signals leads to an aborted T cell activation pathway and the development of unresponsiveness to the specific antigen, or anergy. (See, e.g., Harding, F. A., J. G. McArthur, J. A. Gross., D. M. Raulet, and J. P. Allison, (1992). xe2x80x9cCD28 Mediated Signalling Co-sbmulates Murine T Cells and Prevents Induction of Anergy in T Cell Clones.xe2x80x9d Nature, 356:607-609; Gimmi, C. D., G. J. Freeman, J. G. Gribben, G. Gray, and L. M. Nadler, (1993). xe2x80x9cHuman T-Cell Clonal Anergy is Induced by Antigen Presentation in the Absence of B7 Costimulation.xe2x80x9d Proc. Natl. Acad. Sci., 90:6586-6590; Tan, P., C. Anasefti, J. A. Hansen, J. Melrose, M. Brunvand, J. Bradshaw, J. A. Ledbetter, and P. S. Linsley, (1993), xe2x80x9cInduction of Alloantigen-specific Hyporesponsiveness in Human T Lymphocytes by Blocking Interaction of CD28 with Its Natural Ligand B7/BB1.xe2x80x9d J. Exp. Med., 177:165-173). Achievement of in vivo tolerance constitutes a mechanism for immunosuppression and a viable therapy for organ transplant rejection and for the treatment of autoimmune diseases. This has been achieved in experimental models following the administration of CTLA4-Ig (Lenschow, D. J., Y. Zeng, R. J. Thistlethwaite, A. Montag, W. Brady, M. G. Gibson, P. S. Linsley, and J. A. Bluestone, (1992), xe2x80x9cLong-Term Survival of Xenogeneic Pancreatic Islet Grafts Induced by CTLA-4Ig.xe2x80x9d Science, 257:789-795).
The molecules B7.1 and B7.2 can bind to either CD28 or CTLA-4, although B7.1 binds to CD28 with a Kd of 200 Nm and to CTLA-4 with a 20-fold higher affinity (Linsley, P. S., E. A. Clark, and J. A. Ledbetter, (1990), xe2x80x9cT-Cell Antigen CD28 Mediates Adhesion with B Cells by Interacting with Activation Antigen B7/BB-1.xe2x80x9d Proc. Natl. Acad. Sci., 87:5031-5035; Linsley et al, (1993), xe2x80x9cThe Role of the CD28 receptor during T cell responses to antigen,xe2x80x9d Annu. Rev. Immunol., 11:191-192; Linesley et al, (1993), xe2x80x9cCD28 Engagement by B7/BB-1 Induces Transient Down-Regulation of CD28 Synthesis and Prolonged Unresponsiveness to CD28 Signaling,xe2x80x9d The Journal of Immunology, 150:3151-3169). B7.2 is expressed on activated B cells and interferon induced monocytes, but not resting B cells (Freeman, G. J., G. S. Gray, C. D. Gimmi, D. B. Lomarrd, L-J. Zhou, M. White, J. D. Fingeroth, J. G. Gribben, and L M. Nadler, (1991). xe2x80x9cStructure, Expression and T Cell Costimulatory Activity of the Murine Homologue of the Human B Lymphocyte Activation Antigen B7,xe2x80x9d J. Exp. Med., 174:625-631). B7.2, on the other hand, is constitutively expressed at very low levels on resting monocytes, dendritic cells and B cells, and its expression is enhanced on activated T cells, NK cells and B lymphocytes (Azuma, M. D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L. L. Lanier, and C. Somoza, xe2x80x9c1993xe2x80x9d, xe2x80x9cB70 Antigen is a Second Ligand for CTLA-4 and CD28,xe2x80x9d Nature, 366:76-79). Although B7.1 and B7.2 can be expressed on the same cell type, their expression on B cells occurs with different kinetics (Lenschow, D. J., G. H. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, G. S. Gray, J. Miller, and J. A. Bluestone, (1993), xe2x80x9cExpression and Functional Significance of an Additional Ligand for CTLA-4,xe2x80x9d Proc. Natl. Acad. Sci., USA, 90:11054-11058; Boussiotis, V. A., G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M. Nadler, (1993), xe2x80x9cActivated Human B Lymphocytes Express Three CTLA-4 Counter-receptors that Co-stimulate T-Cell Activation.xe2x80x9d Proc. Natl. Acad. Sci., USA, 90:11059-11063). Further analysis at the RNA level has demonstrated that B7.2 mRNA is constitutively expressed, whereas B7.1 MRNA is detected 4 hours after activation and initial low levels of B7.1 protein are not detectable until 24 hours after stimulation (Boussiotis, V. A., G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M. Nadler, (1993), xe2x80x9cActivated Human B Lymphocytes Express Three CTLA-4 Counter-receptors that Co-stimulate T-Cell Activation,xe2x80x9d Proc. Natl. Acad. Sci., USA, 90:11059-11063). CTLA-4/CD28 counter receptors, therefore, may be expressed at various times after B Cell activation.
The differential temporal expression of B7.1 and B7.2 suggests that the interaction of these two molecules with CTLA-4 and/or CD28 deliver distinct but related signals to the T cell (LaSalle, J. M., P. J. Tolentino, G. J. Freeman, L. M. Nadler, and D. A. Hafler, (1992), xe2x80x9cCD28 and T Cell Antigen Receptor Signal Transduction Coordinately Regulate Intedeukin 2 Gene Expression In Response to Superantigen Stimulation,xe2x80x9d J. Exp. Med., 176:177-186; Vandenberghe, P., G. J. Freeman, L. M. Nadler, M. C. Fletcher, M. Kamoun, L. A. Turka, J. A. Ledbetter, C. B. Thompson, and C. H. June, (1992), xe2x80x9cAntibody and B7/BB1-mediated Ligation of the CD28 Receptor Induces Tyrosine Phosphorylation in Human T Cells,xe2x80x9d The Journal of Experimental Medicine, 175:951-960). The exact signaling functions of CTLA-4 and CD28 on the T cell are currently unknown (Janeway, C. A., Jr. and K. Bottomly, (1994), xe2x80x9cSignals and Signs for Lymphocyte Responses,xe2x80x9d Cell, 76.275285). However, it is possible that one set of receptors could provide the initial stimulus for T cell activation and the second, a sustained signal to allow further elaboration of the pathway and clonal expansion to take place (Linsley, P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti, and N. K. Damle, (1992), xe2x80x9cCoexpression and Functional Cooperation of CTLA-4 and CD28 on Activated T Lymphocytes,xe2x80x9d J. Exp. Med., 176:1595-1604). The current data supports the two-signal hypothesis proposed by Jenkins and Schwartz (Schwartz, R. H., (1990), xe2x80x9cA Cell Culture Model for T Lymphocyte Clonal Anergy,xe2x80x9d Science, 248:1349; Jenkins, M. K., (1992), xe2x80x9cThe Role of Cell Division in the Induction of Clonal Anergy,xe2x80x9d Immunology Today, 13:69) that both a TCR and co-stimulatory signal are necessary for T cell expansion, lymphokine secretion and the full development of effector function (Greenan, V. and G. Kroemer, (1993), xe2x80x9cMultiple Ways to Cellular Immune Tolerance,xe2x80x9d Immunology Today, 14:573). The failure to deliver the second signal results in the inability of T cells to secrete IL-2 and renders the cell unresponsive to antigen.
Structurally, both B7.1 and B7.2 contain extracellular immunoglobulin superfamily V and C-like domains, a hydrophobic transmembrane region and a cytoplasmic tail (Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J. W. Ng, V. Restivo, Jr., L. A. Lombard, G. S. Gray, and L. M. Nadler, (1993), xe2x80x9cCloning of B7-2: A CTLA-4 Counter-receptor that Co-stimulates Human T Cell Proliferation,xe2x80x9d Science, 262:909). Both B7.1 and B7.2 are heavily glycosylated. B7.1 is a 44-54 kD glycoprotein comprised of a 223 amino acid extracellular domain, a 23 amino acid transmembrane domain, and a 61 amino acid cytoplasmic tail. B7.1 contains 3 potential protein kinase phosphorylation sites. (Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L. L. Lanier, (1993), xe2x80x9cFunctional Expression of B7/BB1 on Activated T Lymphocytes,xe2x80x9d J. Exp. Med., 177:845-850). B7.2 is a 306 amino acid membrane glycoprotein. It consists of a 220 amino acid extracellular region, a 23 amino acid hydrophobic transmembrane domain and a 60 amino acid cytoplasmic tail (Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler, (1989), xe2x80x9cB7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells,xe2x80x9d The Journal of Immunology, 143:2714-2722). Although both B7.1 and B7.2 genes are localized in the same chromosomal region (Freeman, G. J., D. B. Lombard, C. D. Gimmi, S. A. Brod, L Lee, J. C. Laning, D. A. Hafler, M. E. Dorf, G. S. Gray, H. Reiser, C. H. June, C. B. Thompson, and L. M. Nadler, (1992), xe2x80x9cCTLA-4 and CD28 MRNA are Coexpressed in Most T Cells After Activation,xe2x80x9d The Journal of Immunology, 149:3795-3801; Schwartz, R. H., (1992), xe2x80x9cCostimulation of T Lymphocytes: The Role of CD28, CTLA-4, and B7/BB1xe2x80x9d in Selvakumar, A., B. K. Mohanraj, R. L. Eddy, T. B. Shows, P. C. White, C. Perrin, and B. Dupont, (1992), xe2x80x9cGenomic Organization and Chromosomal Location of the Human Gene Encoding the B-Lymphocyte Activation Antigen B7,xe2x80x9d Immunogenetics, 36:175-181), these antigens do not share a high level of homology. The overall homology between B7.1 and B7.2 is 26% and between murine B7.1 and human S7 is 27% (Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L. L. Lanier, (1993), xe2x80x9cFunctional Expression of B7/BB1 on Activated T Lymphocytes,xe2x80x9d J. Exp. Med., 177:845-850; Freeman, G. J., A. S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler, (1989), xe2x80x9cB7, A New Member of the Ig Superfamily with Unique Expression on Activated and Neoplastic B Cells,xe2x80x9d The Journal of Immunology, 143:2714-2722). Although alignment of human B7.1 human B7.2 and murine B.1 sequences shows few stretches of lengthy homology, it is known that all three molecules bind to human CTLA-4 and CD28. Thus, there is most likely a common, or closely homologous region shared by the three molecules that may be either contiguous or conformational. This region may constitute the binding site of the B7.1 and B7.2 molecules to their counter-receptors. Antibodies raised against these epitopes could potentially inhibit the interaction of B7 with its counter-receptor on the T cell. Furthermore, antibodies that cross-reacted with this region on both B7.1 and B7.2 molecules would potentially have practical advantages over antibodies directed against B7.1 or B7.2 separately.
2. Blockade of the B7/CD28 Interaction
Blocking of the B7/CD28 interaction offers the possibility of inducing specific immunosuppression, with potential for generating long lasting antigen-specific therapeutic effects. Antibodies to either B7.1 or B7.2 have been shown to block T cell activation, as measured by the inhibition of IL-2 production in vitro (DeBoer, M., P. Parren, J. Dove, F. Ossendorp, G. van der Horst, and J. Reeder, (1992), xe2x80x9cFunctional Characterization of a Novel Anti-B7 Monoclonal Antibody,xe2x80x9d Eur. Journal of Immunology, 22:3071-3075; Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L. L. Lanier, (1993), xe2x80x9cFunctional Expression of B7/BB1 on Activated T Lymphocytes,xe2x80x9d J. Exp. Med., 177:845-850). However, different antibodies have been shown to vary in their immunosuppressive potency, which may reflect either their affinity or epitope specificity. CTLA-4/lg fusion protein and anti-CD28 Fabs were shown to have similar effects on the down regulation of IL-2 production.
In vivo administration of a soluble CTLA-4/lg fusion protein has been shown to suppress T cell-dependent antibody responses in mice (Linsley, P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti, and N. K. Damle, (1992), xe2x80x9cCoexpression and Functional Cooperation of CTLA-4 and CD28 on Activated T Lymphocytes,xe2x80x9d J. Exp. Med., 176:1595-1604; Lin, H., S. F. Builing, P. S. Linsley, R. O. Wei, C. D. Thompson, and L. A. Turka, (1993), xe2x80x9cLong-term Acceptance of Major Histocompatibility Complex Mismatched Cardiac Allografts Induced by CTLA-4-Ig Plus Donor Specific Transfusion,xe2x80x9dJ. Exp. Med., 178:1801) and, furthermore, larger doses were also able to suppress responses to a second immunization, demonstrating the feasibility of this approach for the treatment of antibody mediated autoimmune disease. In addition, CTLA-4/Ig was able to prevent pancreatic islet cell rejection in mice by directly inhibiting the interaction of T cells and B7.1/B7.2 antigen presenting cells (Lenschow, D. J., G. H. Su, L. A. Zuckerman, N. Nabavi, C. L. Jellis, G. S. Gray, J. Miller, and J. A. Bluestone, (1993), xe2x80x9cExpression and Functional Significance of an Additional Ligand for CTLA-4,xe2x80x9d Proc. Natl. Acad. Sci., USA, 90:11054-11058). In this case, long term donor specific tolerance was achieved.
3. Recombinant Phage Display Technology for Antibody Selection
To date, no monoclonal antibodies which crossreact with both B7.1 and B7.2 have been reported. As noted, such antibodies would potentially be highly desirable as immunosuppressants. Phage display technology is beginning to replace traditional methods for isolating antibodies generated during the immune response, because a much greater percentage of the immune repertoire can be assessed than is possible using traditional methods. This is in part due to PEG fusion inefficiency, chromosomal instability, and the large amount of tissue culture and screening associated with heterohybridoma production. Phage display technology, by contrast, relies on molecular techniques for potentially capturing the entire repertoire of immunoglobulin genes associated with the response to a given antigen.
This technique is described by Barber et al, Proc. Natl. Acad. Sci., USA, 88, 7978-7982, (1991). Essentially, immunoglobulin heavy chain genes are PCR amplified and cloned into a vector containing the gene encoding the minor coat protein of the filamentous phage M13 in such a way that a heavy chain fusion protein is created. The heavy chain fusion protein is incorporated into the M13 phage particle together with the light chain genes as it assembles. Each recombinant phage contains, within its genome, the genes for a different antibody Fab molecule which it displays on its surface. Within these libraries, in excess of 106 different antibodies can be cloned and displayed. The phage library is panned on antigen coated microliter wells, non-specific phage are washed off, and antigen binding phage are eluted. The genome from the antigen-specific clones is isolated and the gene III is excised, so that antibody can be expressed in soluble Fab form for further characterization. Once a single Fab is selected as a potential therapeutic candidate, it may easily be converted to a whole antibody. A previously described expression system for converting Fab sequences to whole antibodies is IDEC""s mammalian expression vector NEOSPLA. This vector contains either human gamma 1 or gamma 4 constant region genes. CHO cells are transfected with the NEOSPLA vectors and after amplification this vector system has been reported to provide very high expression levels ( greater than 30 pg/cell/day) can be achieved.
4. Primatized Antibodies
Another highly efficient means for generating recombinant antibodies is disclosed by Newman, (1992), Biotechnology, 10, 1455-1460. More particularly, this technique results in the generation of primatized antibodies which contain monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. Moreover, this technique is also described in commonly assigned U.S. application Ser. No. 08/379,072, filed on Jan. 25, 1995, which is a continuation of U.S. Ser. No. 07/912,292, filed Jul. 10, 1992, which is a continuation-in-part of U.S. Ser. No. 07/856,281, filed Mar. 23, 1992, which is finally a continuation-in-part of U.S. Ser. No. 07/735,064, filed Jul. 25, 1991. Ser. No. 08/379,072 and the parent application thereof are incorporated by reference in their entirety herein.
This technique modifies antibodies such that they are not antigenically rejected upon administration in humans. This technique relies on immunization of cynomolgus monkeys with human antigens or receptors. This technique was developed to create high affinity monoclonal antibodies directed to human cell surface antigens.
Antibodies generated in this manner have previously been reported to display human effector function, have reduced immunogenicity, and long serum half-life. The technology relies on the fact that despite the fact that cynomolgus monkeys are phylogenetically similar to humans, they still recognize many human proteins as foreign and therefore mount an immune response. Moreover, because the cynomolgus monkeys are phylogenetically close to humans, the antibodies generated in these monkeys have been discovered to have a high degree of amino acid homology to those produced in humans. Indeed, after sequencing macaque immunoglobulin light and heavy chain variable region genes, it was found that the sequence of each gene family was 85-98% homologous to its human counterpart (Newman et al, (1992), Id.). The first antibody generated in this way, an anti-CD4 antibody, was 91-92% homologous to the consensus sequence of human immunoglobulin framework regions. Newman et al, Biotechnology, 10:1458-1460, (1992).
Monoclonal antibodies specific to the human B7 antigen have been previously described in the literature. For example, Weyl et al, Hum. Immunol., 31(4), 271-276, (1991) describe epitope mapping of human monoclonal antibodies against HLA-B-27 using natural and mutated antigenic variants. Also, Toubert et al, Clin. Exp. Immunol., 82(1), 16-20, (1990) describe epitope mapping of an HLA-B27 monoclonal antibody that also reacts with a 35-KD bacterial outer membrane protein. Also, Valle et al, Immunol., 69(4), 531-535, (1990) describe a monoclonal antibody of the IgG1 subclass which recognizes the B7 antigen expressed in activated B cells and HTLV-1-transformed T cells. Further, Toubert et al, J. Immunol., 141(7), 2503-9, (1988) describe epitope mapping of HLA-B27 and HLA-B7 antigens using intradomain recombinants constructed by making hybrid genes between these two alleles in E. coli. 
High expression of B7 antigen has been correlated to autoimmune diseases by some researchers. For example, Ionesco-Tirgoviste et al, Med. Interre, 24(1), 11-17, (1986) report increased B7 antigen expression in type 1 insulin-dependent diabetes. Also, the involvement of B7 antigen expression on dermal dendritic cells obtained from psoriasis patients has been reported. (Nestle et al, J. Clin. Invest., 94(1), 202-209, (1994)).
Further, the inhibition of anti-HLA-B7 alloreactive CTL using affinity-purified soluble HLA-B7 has been reported in the literature. (Zavazava et al, Transplantation, 51(4), 838-42, (1991)). Further, the use of B7 receptor soluble ligand, CTLA-4-Ig to block B7 activity (See, e.g., Lenschow et al, Science, 257, 789, 7955 (1992)) in animal models and a B7-1-Ig fusion protein capable of inhibiting B7 has been reported.
An object of the invention is to produce and identify novel macaque antibodies to human B7 antigen, more specifically to human B7.1 antigen and/or human B7.2 antigen.
More specifically, it is an object of the present invention to produce and identify novel macaque antibodies to human B7 antigen, i.e., human B7.1 and human B7.2 antigen by screening of phage display libraries and/or monkey heterohybridomas using B lymphocytes obtained from human B7 antigen, i.e., human B7.1 or B7.2 antigen immunized monkeys.
It is another specific object of the invention to provide anti-B7 monkey monoclonal antibodies and primatized forms thereof which specifically bind human B7.1 and/or B7.2 antigen which inhibit the:B7/CD86 pathway and B7 stimulation of activated T cells, thereby inhibiting IL-2 production and T cell proliferation and functioning as effective immunosuppressants.
It is another object of the invention to provide anti-human B7.1 and anti-human B7.2 monkey monoclonal antibodies and primatized forms thereof which inhibit antigen driven responses in donor spleen cell cultures, e.g., antigen specific IgG responses, IL-2 production and cell proliferation.
It is another specific object of the invention to identify particular monkey monoclonal antibodies specific to human B7.1 and human B7.2 antigen and primatized forms thereof having advantageous properties, i.e., affinity, immunosuppressive activity, which are useful as therapeutics. More specifically, these monkey antibodies and primatized forms thereof are to be used, e.g., as immunosuppressants, i.e., to block antigen driven immune responses, to treat autoimmune diseases such as psoriasis, rheumatoid arthritis, systemic erythematosus (SLE), type 1 diabetes mellitus, idiopathic thrombocytopenia purpura (ITP), and to prevent organ rejection.
It is another object of the invention to provide pharmaceutical compositions containing one or more monkey monoclonal antibodies specific to human B7 antigen, i.e., human B7.1 and/or human B7.2 antigen, or primatized forms thereof, and a pharmaceutically acceptable carrier or excipient. These compositions will be used, e.g., as immunosuppressants to treat autoimmune diseases, e.g., idiopathic thrombocytopenia purpura (ITP) and systemic lupus erythematosus (SLE), to block antigen driven immune responses, and to prevent organ rejection in transplant recipients.
It is another object of the invention to provide novel methods of therapy by administration of therapeutically effective amounts of one or more monkey or primatized monoclonal antibodies which specifically bind to B7 antigen, i.e., human B7.1 and/or B7.2 antigens. Such therapeutic methods are useful for treatment of diseases treatable by inhibition of the B7:CD28 pathway e.g., autoimmune diseases such as idiopathic thrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type 1 diabetes mellitus, psoriasis, rheumatoid arthritis, multiple sclerosis, aplastic anemia, as well as for preventing rejection in transplantation subjects.
It is still another object of the invention to provide transfectants, e.g., CHO cells, which express at least the variable heavy and light domains of monkey monoclonal antibodies specific to the human B7.1 and/or B7.2 antigen.
It is another object of the invention to provide nucleic acid sequences which encode the variable heavy and/or light domains of monkey monoclonal antibodies specific to human B7.1 and/or human B7.2 antigen, and expression vectors which provide for the expression of primatized antibodies containing these nucleic acid sequences.
The following terms are defined so that the invention may be more clearly understood.
Depleting antibodyxe2x80x94an antibody which kills activated B cells or other antigen presenting cells.
Non-depleting antibodyxe2x80x94an antibody which blocks the co-stimulatory action of B7 and T cell activating ligands CD28 and CTLA-4. Thus, it anergizes but does not eliminate the antigen presenting cell.
Primatized antibodyxe2x80x94a recombinant antibody which has been engineered to contain the variable heavy and light domains of a monkey antibody, in particular, a cynomolgus monkey antibody, and which contains human constant domain sequences, preferably the human immunoglobulin gamma 1 or gamma 4 constant domain (or PE variant). The preparation of such antibodies is described in Newman et al, (1992), xe2x80x9cPrimatization of Recombinant Antibodies for Immunotherapy of Human Diseases: A Macaque/Human Chimeric Antibody Against Human CDH, Biotechnology, 10:1458-1460; also in commonly assigned Ser. No. 08/379,072 both of which are incorporated by reference in their entirety herein. These antibodies have been reported to exhibit a high degree of homology to human antibodies, i.e., 85-98%, display human effector functions, have reduced immunogenicity, and may exhibit high affinity to human antigens.
B7 antigensxe2x80x94B7 antigens in this application include, e.g., human B7, B7.1 and B7.2 antigens. These antigens bind to CD28 and/or CTLA-4. These antigens have a co-stimulatory role in T cell activation. Also, these B7 antigens all contain extracellular immunoglobulin superfamily V and C-like domains, a hydrophobic transmembrane region and a cytoplasmic tail. (See, Freeman et al, Science, 262:909, (1993)), and are heavily glycosylated.
Anti-B7 antibodiesxe2x80x94Antibodies, preferably monkey monoclonal antibodies or primatized forms thereof, which specifically bind human B7 antigens, e.g., human B7.1 and/or B7.2 antigen with a sufficient affinity to block the B7:CD28 interaction and thereby induce immunosuppression.